Among bioactive substances or environmental pollutants such as natural products, toxins, hormones, or agricultural chemicals, numerous substances act in ultratrace amounts. Accordingly, instrumental analytical methods capable of performing high-sensitivity analysis have conventionally been widely used for qualitative and quantitative measurement of these substances. However, instrumental analytical methods are poor in specificity, require excessive time for analysis including pretreatment of samples, and are troublesome in operation. Thus instrumental analytical methods are inconvenient for the purpose of rapid and convenient measurements that have been required in recent years. Meanwhile, immunoassays are highly specific and much easier in terms of operation than instrumental analytical methods. Therefore immunoassays have gradually spread in the field of measurement of bioactive substances and environmental pollutants. However, conventional immunoassays such as enzyme immunoassays using 96-well plates and latex agglutination assays do not always provide satisfactory rapidness and convenience for measurement or detection sensitivity.
Another need expected to be enabled is as follows. Achievement of higher sensitivity of tests that currently use relatively invasive samples such as swabs and blood makes it possible to detect very small amounts of analytes contained in relatively low-invasive samples such as snot, mouth wash, and urine. Thus, less burdensome tests of patients can be realized.
In recent years, test kits using an immunochromatography method (hereinafter referred to as an immunochromatography kit) have been used more often in examination of infections that require particularly rapid diagnosis. According to the spread of these kits, patients with infections can be identified by a rapid and convenient method, and subsequent diagnosis and therapy can be conducted immediately and accurately. For example, in an immunochromatography method using the sandwich method, a labeled second antibody capable of specifically binding to an analyte (for example, an antigen), and a sample solution which may possibly contain the analyte are developed on an insoluble thin film-shaped support (for example, a glass fiber membrane, a nylon membrane, or a cellulose membrane) on which a first antibody capable of specifically binding to the analyte has been immobilized in a specific region. As a result, an immune complex with the analyte is formed at the region of the insoluble thin film-shaped carrier, on which region the first antibody has been immobilized. The analyte can be measured by detecting a signal such as color development or coloring of a label. The label to be used herein may be, for example, a protein such as an enzyme, colored latex particles, metal colloids, or carbon particles.
The immunochromatography method requires neither massive facilities nor instruments for determination and measurement. Furthermore, the immunochromatography method is simple in operation and promptly gives measurement results by introducing a sample solution dropwise which may possibly contain an analyte and leaving it for approximately 5 to 10 minutes. For this reason, this technique is used widely as a convenient, rapid, and highly specific method for determination and measurement in many scenarios, such as for clinical examination in hospitals and in assays in laboratories.
Among bioactive substances or environmental pollutants such as natural products, toxins, hormones, and agricultural chemicals, and specimens of early infection stage of virus disease, many substances exert effects in ultratrace amounts that are undetectable by conventional common immunochromatography methods. Therefore, there are demands for development of rapid, convenient, and highly sensitive immunochromatography methods for such substances.
JP Patent Publication (Kokai) No. 2002-202307 A discloses that gold colloids were amplified using a silver sensitizer, “Silver Enhancing Kit (Cat. SEKB250); produced by British BioCell International.” However, the relevant method requires 10 or more minutes for amplification, lacking the rapidness that is a characteristic of immunochromatography methods. Furthermore, JP Patent Publication (Kohyo) No. 10-513263 A (1998) discloses that gold colloids are amplified using an optical microscope silver enhancing kit (SELK15; produced by British BioCell International). Since the amount of labeling substance at detection site can not be specified by the disclosure of JP Patent Publication (Kohyo) No. 10-513263 A (1998), a makeup experiment was carried out using an amplification kit for photomicroscope, SEKL15 (British Biocell International), wherein the amount of the labeling substance at the detection site was changed. As a result, although amplification was confirmed, the particle size was only 350 nm. From this result, it was considered that a particle of 1 nm which cannot be observed visually is amplified and very small amount of analyte cannot be detected in JP Patent Publication (Kohyo) No. 10-513263 A (1998).